Aggrecan is a major extracellular component of articular cartilage. It is a proteoglycan responsible for providing cartilage with its -mechanical properties of compressibility and elasticity. The loss of aggrecan has been implicated in the degradation of articular cartilage in arthritic diseases such as osteoarthritis.
Osteoarthritis is a debilitating disease which affects at least 30 million Americans (MacLean et al., J Rheumatol 25:2213–2218 (1998)). Osteoarthritis can severely reduce quality of life due to degradation of articular cartilage and the resulting chronic pain. An early and important characteristic of the osteoarthritic process is loss of aggrecan from the extracellular matrix (Brandt and Mankin, “Pathogenesis of Osteoarthritis,” Textbook of Rheumatology, WB Saunders Company, Philadelphia, Pa., pgs. 1355–1373 (1993)). The large, sugar-containing portion of aggrecan is thereby lost from the extra-cellular matrix, resulting in deficiencies in the biomechanical characteristics of the cartilage.
A proteolytic activity termed “aggrecanase” is believed to be responsible for the cleavage of aggrecan, thereby having a role in cartilage degradation associated with osteoarthritis and inflammatory joint disease. Research has been conducted to identify the enzymes responsible for the degradation of aggrecan in human osteoarthritic cartilage. Aggrecan contains two N-terminal globular domains, G1 and G2, separated by a proteolytically sensitive interglobular domain, followed by a glycosaminoglycan attachment region and a C-terminal globular domain, G3. At least two enzymatic cleavage sites have been identified within the interglobular domain of aggrecan. One enzymatic cleavage site within the interglobular domain of aggrecan (Asn341-Phe342) has been observed to be cleaved by several known metalloproteases. Flannery et al., J Biol Chem 267:1008–1014 (1992); Fosang et al., Biochemical J. 304:347–351 (1994). Cleavage at a second aggrecan cleavage site within aggrecan (Glu373-Ala374) due to IL-1 induced cartilage aggrecan cleavage results in the generation of an aggrecan fragment found in human synovial fluid (Sandy et al., J Clin Invest 89:1512–1516 (1992); Lohmander et al., Arthritis Rheum 36: 1214–1222 (1993); Sandy et al., J Biol Chem 266: 8683–8685 (1991)). Aggrecan cleavage at (Glu373-Ala374) has been attributed to aggrecanase activity (Sandy et al., J Clin Invest 89:1512–1516 (1992). This Glu373-Ala374 cleavage site will be referred to as the aggrecanase cleavage site.
Recently, identification of two enzymes, aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS-11) within the “A Disintegrin and Metalloproteinase with Thrombospondin motifs” (ADAMTS) family, have been identified which are synthesized by IL-1 stimulated cartilage and cleave aggrecan at the Glu373-Ala374 site (Tortorella et al., Science 284:1664–1666 (1999); Abbaszade et al., J Biol Chem 274: 23443–23450 (1999)). Aggrecanase-1 is reported to include at least six domains: signal; propeptide; catalytic; disintegrin; TSP type-1 motif and C-terminal. Aggrecanase-2 is also a multidomain protein. It is reported to have a signal sequence; a prodomain; a metalloproteinase domain; a disintegrin domain and a spacer domain between a TSP motif and a TSP sub motif in the C-terminal of the protein. It was generally believed that the TSP domains and the spacer domain are critical for substrate recognition. Specifically, Tortorella et al. reported that “[T]his region may serve to bind aggrecanase-1 to the glycosaminoglycans of the aggrecan substrate.” See Tortorella et al., Science 284:1664–1666 (1999).
It is contemplated that there are other, related enzymes in the ADAMTS family which are capable of cleaving aggrecan at the Glu373-Ala374 bond and could contribute to aggrecan cleavage in osteoarthritis. It is possible that these enzymes could be synthesized by osteoarthritic human articular cartilage. However, it has been difficult to develop inhibitors and treatment therapies to treat diseases that involve aggrecan cleavage because aggrecanases have been difficult to isolate and purify in large amounts due to poor stability of these molecules and generally low expression levels. Therefore, there is a need to identify novel forms of aggrecanases and further develop ways to isolate and purify aggrecanase proteins in large amounts in order to investigate their role in disease states and also to develop therapies and compositions to treat diseases involving aggrecan cleavage.